重组C-反应蛋白的纯化与肽标记的同位素稀释质谱测定  

The Purification and Quantification of Recombinant C-Reaction Protein by PC-IDMS

作　　者：张春鹂 宋德伟[1] 孙巍[1] 刘钰[2] 肖鹏[2] 李红梅[2] 

机构地区：[1]北京化工大学,北京100029 [2]中国计量科学研究院,北京100029

出　　处：《生命科学仪器》2016年第1期47-50,共4页Life Science Instruments

基　　金：科技部重大项目863计划(2011AA02A111);科技部基础性工作专项(2011FY130100);国家科技支撑计划(2013BAK10B01);质检公益性行业专项(201410234)资助

摘　　要：本文依次采用Immobilized p-Aminophenyl Phosphoryl Choline Gel亲和层析柱和Superdex 75 10/300 GL分子筛层析柱分离纯化HEK293细胞重组C-反应蛋白(C-reactive protein,CRP),对其进行了凝胶电泳、酶切、MALDI-TOF-MS鉴定,并利用蛋白质酶解-同位素稀释质谱法对其进行准确定量。结果表明,重组得到的样品符合CRP结构特征,液相纯度为96.527%,蛋白质酶解-同位素稀释质谱法定量的结果为3.32mg/g,扩展不确定度为0.19mg/g(k=2)。本文建立的蛋白质酶解-同位素稀释质谱法不受样品中肽类/蛋白类杂质的影响,提高了C-反应蛋白含量测定的准确度和精密度,定量结果可溯源至SI单位,为C-反应蛋白标准物质的研制奠定了基础。Recombinant C-reaction protein （CRP） was isolated and purified from human 293 cells （HEK293） by Immobilized p-Aminophenyl Phosphoryl Choline Gel affinity column and Superdex 75 10~300 GL molecular sieve column orderly. And then it was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）, trypsin digestion and MALDI- TOF-MS. Finally, a new method for accurate quantification of CRP was established by protein cleavage isotope dilution mass spectrometry （PC-IDMS）. Results showed that the sample obtained was in accord with the structure characteristic of CRP and the purity determined by high performance liquid chromatographic （HPLC） was 96.527%. The content of purified recombinant CRP was calculated to be 3.32mg/g with the uncertainty of O.19mg/g （k=2） based on PC-IDMS. The method established by PC-IDMS will not be affected by peptide/protein impurities in the sample, particularly, and the measure result can trace to SI units. In conclusion, application of this method for quantification of C-reaction protein will improve the accuracy and precision and lay the foundation for the development of C-reaction protein reference material.

关 键 词：C-反应蛋白 亲和层析法 肽类 同位素稀释质谱法 

分 类 号：O657[理学—分析化学]